阅读说明：本技术 用于治疗糖原贮积病的方法和组合物 (Methods and compositions for treating glycogen storage disease ) 是由 克里斯托弗·蒂珀 凯利·里德·克拉克 塞缪尔·沃兹沃思 于 2019-12-18 设计创作，主要内容包括：本发明提供了多种新颖腺相关病毒(AAV)载体,用于治疗Ia型糖原贮积病(GSD-Ia)的基因疗法应用。本文披露了许多掺入有经修饰的G6PC启动子/增强子序列的重组核酸分子、载体和重组AAV。当从各种宿主细胞平台表达时,使用所述经修饰的G6PC启动子/增强子序列导致增强的AAV产量和质量。本文还提供包含本发明的新颖AAV的组合物和使用所述组合物治疗GSD-Ia的方法。(The present invention provides novel adeno-associated virus (AAV) vectors for gene therapy applications in the treatment of glycogen storage disease type Ia (GSD-Ia). Disclosed herein are a number of recombinant nucleic acid molecules, vectors, and recombinant AAVs that incorporate modified G6PC promoter/enhancer sequences. The use of the modified G6PC promoter/enhancer sequence results in enhanced AAV yield and quality when expressed from various host cell platforms. Also provided herein are compositions comprising the novel AAVs of the invention and methods of using the compositions in the treatment of GSD-Ia.)

1. A recombinant nucleic acid molecule comprising a modified G6PC promoter/enhancer (GPE) sequence, wherein the modified GPE lacks one or more sequences that are at least 80% identical to Alu elements.

2. The recombinant nucleic acid molecule of claim 1, wherein said Alu element is selected from the group consisting of contiguous nucleotides 1079-1272(Alu-1), 1633-1968(Alu-2) and 2140-2495(Alu-3) of SEQ ID NO. 6.

3. The recombinant nucleic acid molecule of claim 1 or 2, wherein the modified GPE has a sequence that is 80% identical to contiguous nucleotide 146 and 212380 of SEQ ID NO 1, or to any one of SEQ ID NO 7, 8, 9, 10, 11 or 12.

4.A recombinant nucleic acid molecule comprising a modified G6PC promoter/enhancer (GPE) sequence and a G6P enzyme-a coding sequence, wherein the modified GPE is capable of directing expression of the G6P enzyme-a coding sequence.

5. The recombinant nucleic acid molecule of claim 4, wherein the modified GPE lacks one or more sequences that are at least 80% identical to an Alu element selected from the group consisting of contiguous nucleotides 1079-1272(Alu-1), 1633-1968(Alu-2), and 2140-2495(Alu-3) of SEQ ID NO: 6.

6. The recombinant nucleic acid molecule of claim 4 or 5, wherein the modified GPE has a sequence that is 90% identical to contiguous nucleotide 146 and 212380 of SEQ ID NO 1, or to any one of SEQ ID NO 7, 8, 9, 10, 11 or 12.

7. The recombinant nucleic acid molecule of any one of claims 4-6, wherein the G6P enzyme-a coding sequence comprises the same sequence as SEQ ID No. 3 or SEQ ID No. 4.

8. The recombinant nucleic acid molecule of any one of claims 4-7, wherein said recombinant nucleic acid molecule further comprises a polyadenylation (poly A) signal sequence.

9. The recombinant nucleic acid molecule of claim 8, wherein said polyadenylation signal sequence is the SV40 polyA signal sequence.

10. The recombinant nucleic acid molecule of any one of claims 4-9, wherein the recombinant nucleic acid molecule further comprises an intron.

11. The recombinant nucleic acid molecule of any one of claims 4-10, wherein the recombinant nucleic acid molecule comprises SEQ ID No. 1 or SEQ ID No. 2.

12. A recombinant vector comprising the recombinant nucleic acid molecule of any one of claims 4-11.

13. The vector of claim 12, wherein the vector is an adeno-associated virus (AAV) vector.

14. The vector of claim 13, wherein the AAV vector is an AAV serotype 8(AAV8) vector.

15. An isolated host cell comprising the recombinant nucleic acid molecule of any one of claims 1-11.

16. An isolated host cell comprising the recombinant vector of any one of claims 12-14.

17. A method of increasing rAAV production, the method comprising delivering the recombinant vector of claim 13 or 14 to a eukaryotic host cell culture and harvesting the rAAV from the eukaryotic cell culture.

18. A recombinant AAV (raav) comprising a recombinant nucleic acid molecule according to any one of claims 4-11.

19. The rAAV according to claim 18, wherein the rAAV is a rAAV 8.

20. A composition comprising the rAAV according to claim 18 or 19, and a pharmaceutically acceptable carrier.

21. A method of treating glycogen storage disease type la (GSD-Ia) in a human subject, the method comprising administering to the human subject a therapeutically effective amount of a rAAV according to any one of claims 18-20, or a composition thereof.

22. The method of claim 21, wherein the rAAV is administered intravenously.

23. The method of claim 21 or 22, wherein the rAAV is administered in an approximate manner1x10¹¹To about 1x10¹⁴Single Genomic Copy (GC)/kg dose.

24. The method of claim 23, wherein the rAAV is administered at about 1x10¹²To about 1x10¹³GC/kg dose was administered.

25. The method of any one of claims 21-24, wherein administering the rAAV comprises administering a single dose of rAAV.

26. The method of any one of claims 21-24, wherein administering the rAAV comprises administering multiple doses of rAAV.

27. A recombinant adeno-associated virus (rAAV) for use in the treatment of GSD-Ia, the rAAV comprising an AAV capsid and packaged therein a vector genome comprising:

(a) AAV 5' Inverted Terminal Repeat (ITR) sequences;

(b) a promoter/enhancer sequence comprising contiguous nucleotides 146-2123 of SEQ ID NO. 1;

(c) a coding sequence encoding glucose-6-phosphatase alpha (G6P enzyme-alpha); and

(d)AAV 3’ITR。

28. the rAAV according to claim 27, wherein the G6P enzyme-a comprises an amino acid sequence that is 590% identical to SEQ ID NO.

29. The rAAV according to claim 27, wherein the amino acid sequence of the G6P enzyme-a is identical to SEQ ID No. 5.

30. The rAAV according to any one of claims 27-29, wherein the coding sequence of (c) is at least 90% identical to SEQ ID No. 3 or SEQ ID No. 4.

31. The rAAV according to any one of claims 27-30, wherein the AAV capsid is an AAV8 capsid.

32. The rAAV according to any one of claims 27-31, wherein the vector genome further comprises a polyadenylation (poly a) signal sequence.

33. The rAAV according to claim 32, wherein the polyadenylation signal sequence is an SV40 polya signal sequence.

34. The rAAV according to any one of claims 27-33, wherein the vector genome further comprises an intron.

35. A composition comprising the rAAV according to any one of claims 27-34, and a pharmaceutically acceptable carrier.

36. A method of treating glycogen storage disease type la (GSD-Ia) in a human subject, the method comprising administering to the human subject a therapeutically effective amount of the rAAV according to any one of claims 27-34 or the composition thereof according to claim 35.

37. The method of claim 36, wherein the rAAV is administered intravenously.

38. The method of claim 36 or 37, wherein the rAAV is administered at about 1x10¹¹To about 1x10¹⁴Single Genomic Copy (GC)/kg dose.

39. The method of claim 38, wherein the rAAV is administered at about 1x10¹²To about 1x10¹³GC/kg dose was administered.

40. The method of any one of claims 36-39, wherein administering the rAAV comprises administering a single dose of the rAAV.

41. The method of any one of claims 36-39, wherein administering the rAAV comprises administering multiple doses of the rAAV.

Technical Field

The present application relates generally to viral vectors for use in the treatment of glycogen storage diseases, such as glycogen storage disease type Ia, and more particularly to adeno-associated viral vectors.

Background

Glycogen storage disease type Ia (also known as GSD-Ia or liver glycogen accumulation (von girerke disease)) is an inherited disorder caused by glycogen accumulation in cells of the body. Glycogen accumulation in certain organs and tissues, particularly in the liver, kidneys and small intestine, impairs their ability to function properly. GSD-Ia usually occurs in the first year after birth, with severe hypoglycemia and hepatomegaly caused by glycogen accumulation. Affected individuals exhibit growth retardation, delayed puberty, lactic acidemia, hyperlipidemia, hyperuricemia, and a high incidence of hepatic adenomas in adults. See Lei et al, 1993, Science [ Science ]262: 580-3.

GSD-Ia is a rare orphan genetic disease caused by a deficiency in active glucose-6-phosphatase-alpha (G6P enzyme-alpha), a key enzyme involved in maintaining glucose homeostasis. The G6P enzyme- α is encoded by the G6PC gene and catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate in the final step of glycogenolysis and gluconeogenesis. To date, more than 80 mutations have been identified that lead to G6P enzyme- α deficiency and the associated development of GSD-Ia. See Chou et al, 2010, Nat Rev Endocrinol [ Nature review Endocrinol ]6(12): 676-88.

GSD-Ia is currently not curable and the current standard of care for patients is dietary supplementation. Dietary strategies, if strictly followed, generally promote normal growth and pubertal development, but dietary therapy does not completely prevent the occurrence of hyperlipidemia, hyperuricemia, lactic acidemia and liver fat accumulation. See Rake et al, 2002, Eur J Pediatr [ European journal of pediatric surgery ]161 supplement 1: S20-34.

Gene therapy approaches using recombinant adeno-associated virus (AAV) carrying G6P enzyme- α have been explored to manage GSD-Ia. See, for example, U.S. patent No. 9,644,216 and U.S. patent publication No. 2017/0362670. However, to use AAV vectors for human gene therapy, it is crucial to develop robust, reliable and scalable vector production methods. The inventors have found that modifying the promoter/enhancer region of the G6PC gene to remove certain sequences (described herein as "Alu elements") significantly improves rAAV yield and quality when expressed from various host cell platforms.

Disclosure of Invention

The present invention provides methods and compositions for treating glycogen storage disease. More specifically, provided herein are recombinant nucleic acid molecules, adeno-associated virus (AAV) vectors, and recombinant adeno-associated virus (rAAV) that can be used in gene therapy applications to treat GSD-Ia.

In one aspect, the application relates to a recombinant nucleic acid molecule comprising a modified G6PC promoter/enhancer (GPE) sequence, wherein the modified GPE lacks one or more sequences that are at least 80% identical to Alu elements. In some embodiments, the Alu element is selected from the group consisting of contiguous nucleotides 1079-1272(Alu-1), 1633-1968(Alu-2) and 2140-2495(Alu-3) of SEQ ID NO: 6. In some embodiments, the modified GPE has a sequence that is identical to contiguous nucleotide 146-212380% (e.g., 80%, 85%, 90%, 95%, or 100%) of SEQ ID NO: 1: or a sequence identical to SEQ ID NO 7, 8, 9, 10, 11, or 1280% (, e.g., 80%, 85%, 90%, 95%, or 100%).

In another aspect, the application relates to a recombinant nucleic acid molecule comprising a modified G6PC promoter/enhancer (GPE) sequence described herein and a G6P enzyme-a coding sequence, wherein the modified GPE is capable of directing expression of the G6P enzyme-a coding sequence. In some embodiments, the G6P enzyme- α coding sequence comprises a sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 100%) identical to SEQ ID No. 3 or SEQ ID No. 4. In some embodiments, the recombinant nucleic acid molecule further comprises a polyadenylation (poly A) signal sequence, such as the SV40 poly A signal sequence (SEQ ID NO: 14). In some embodiments, the recombinant nucleic acid molecule further comprises an intron (SEQ ID NO: 13). In some embodiments, the recombinant nucleic acid molecule comprises SEQ ID NO 1 or SEQ ID NO 2.

In another aspect, the present application relates to a recombinant vector comprising a recombinant nucleic acid molecule described herein. In some embodiments, the vector is an adeno-associated virus (AAV) vector, e.g., an AAV vector of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or rh10 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or rh 10). In exemplary embodiments, the AAV vector is an AAV serotype 8(AAV8) vector. Further provided are host cells comprising the recombinant nucleic acid molecules or recombinant vectors disclosed herein. In particular embodiments, the host cell may be suitable for propagation of AAV.

In another aspect, the application relates to a method of increasing rAAV production, the method comprising delivering an AAV vector described herein to a host cell culture and harvesting rAAV from the cell culture. In some embodiments, the host cell culture is a eukaryotic host cell culture.

Also provided herein are rAAV comprising a recombinant nucleic acid molecule or AAV vector disclosed herein. In some embodiments, the application relates to rAAV for use in the treatment of GSD-Ia, as well as rAAV comprising an AAV capsid and an AAV vector genome packaged therein, the AAV vector genome comprising an AAV 5' Inverted Terminal Repeat (ITR) sequence; a modified GPE sequence disclosed herein; a coding sequence encoding glucose-6-phosphatase α (G6P enzyme- α) or an active fragment or variant thereof; and AAV 3' ITR sequences. In some exemplary embodiments, the AAV capsid is an AAV8 capsid. In some embodiments, the vector genome comprises 5 'and 3' ITR sequences identical to SEQ ID NO 15. In some embodiments, the vector genome comprises a modified GPE comprising contiguous nucleotides 146 and 2123 of SEQ ID NO: 1. In some embodiments, the vector genome further comprises a polyadenylation (poly A) signal sequence, e.g., the SV40 poly A signal sequence (SEQ ID NO: 14). In some embodiments, the vector genome further comprises an intron (SEQ ID NO: 13). In some embodiments, the G6P enzyme-a comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 100%) identical to SEQ ID No. 5. In some embodiments, the amino acid sequence of the G6P enzyme- α comprises SEQ ID NO 5. In some embodiments, the amino acid sequence of the G6P enzyme- α consists of SEQ ID No. 5. In some embodiments, the coding sequence for the G6P enzyme- α is at least 80% (e.g., 80%, 85%, 90%, 95%, or 100%) identical to SEQ ID No. 3 or SEQ ID No. 4. In some exemplary embodiments, the vector genome comprises the same nucleic acid sequence as SEQ ID NO 1 or 2.

The present application further relates to pharmaceutical compositions comprising the rAAV of the invention. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is formulated for subcutaneous, intramuscular, intradermal, intraperitoneal, or intravenous administration. In an exemplary embodiment, the pharmaceutical composition is formulated for intravenous administration.

In yet another aspect, the application relates to a method of treating glycogen storage disease type Ia (GSD-Ia) in a human subject, the method comprising administering to the human subject a therapeutically effective amount of a rAAV disclosed herein. In some embodiments, the rAAV is administered subcutaneously, intramuscularly, intradermally, intraperitoneallyOr intravenous administration. In exemplary embodiments, the rAAV is administered intravenously. In some embodiments, the rAAV is administered at about 1x10¹¹To about 1x10¹⁴Single Genomic Copy (GC)/kg dose. In further embodiments, the rAAV is administered at about 1x10¹²To about 1x10¹³Single Genomic Copy (GC)/kg dose. In some embodiments, a single dose of rAAV is administered. In other embodiments, multiple doses of rAAV are administered.

These and other aspects and features of the present invention are described in the following sections of this application.

Drawings

The present invention may be more fully understood with reference to the following drawings.

FIG. 1A is a schematic of the G6PC expression cassette surrounded by two AAV2 inverted terminal repeats (ITR, SEQ ID NO:15) and comprising GPE, introns, codon optimized human G6PC gene (hG6PCco) and SV40 late poly A tail. Abbreviations used: the GPE-G6P enzyme promoter/enhancer region; the hG6 PCco-human glucose-6-phosphatase coding region (codon optimized); ITR-inverted terminal repeats; SV40L pA-SV40 late polyadenylation signal; UTR-untranslated region. FIG. 1B is a schematic of the G6PC expression cassette containing wild-type (DTC161) or modified GPE (DTC 175 containing the G6PC expression cassette, said G6PC expression cassette containing GPE in which the Alu-1 and Alu-2 sequences are deleted; DTC176 containing the G6PC expression cassette, said G6PC expression cassette containing GPE in which all three Alu elements are present but the orientation of the Alu-1 and Alu-2 sequences is reversed; DTC177 containing the G6PC expression cassette, said G6PC expression cassette containing GPE in which the Alu-1, Alu-2 and Alu-3 sequences are deleted; DTC178 containing the G6PC expression cassette, said G6PC expression cassette containing GPE in which the Alu-3 sequences are deleted; and DTC179 containing the G6PC expression cassette, said G6PC expression cassette containing GPE in which all three Alu elements are present but the Alu-3 sequence is reversed).

Fig. 2 is a schematic of an exemplary AAV vector (DTC161) in which various key elements are shown. The carrier is characterized as follows:

FIG. 3 is a schematic representation of pAAV2-8.KanR (p2123-FH) AAV Rep/Cap plasmids that provide Rep and Cap functions in packaging rAAV when co-transfected into a host cell with an AAV vector.

FIG. 4 is a schematic representation of the pAdDeltaF6(Kan) adenovirus helper plasmid for rAAV production when co-transfected into a host cell with an AAV vector and a Rep/Cap plasmid.

Fig. 5 is a bar graph showing rAAV titers produced from host cells following transfection with various AAV vectors. Three tests were performed under each condition and the standard deviation is shown. Denotes P <0.05 compared to DTC 161.

Figure 6 is a graph showing rAAV titers produced plotted as a function of vector genome size.

Fig. 7 is an image of an agarose gel when full length DNA is isolated from raavs of the control viral vector, DTC161, DTC175, DTC176, DTC177, DTC178 and DTC179 and subjected to agarose gel electrophoresis, which shows bands of released DNA, which assesses the ability of the capsid to degrade and release packaged DNA. The total viral DNA is between 3.8kb and 5 kb. "" indicates the complete genome of full length DNA isolated from control viral vectors after capsid degradation and treatment with Sodium Dodecyl Sulfate (SDS).

Fig. 8A is a graph showing analytical ultracentrifugation traces of particle density from DTC161 vector preparations produced in HEK293 cells. FIG. 8B is a graph showing analytical ultracentrifugation traces from particle density of DTC177 vector (represented by SEQ ID NO: 1) preparations produced in HEK293 cells. Abbreviations used: RI-refractive index.

Fig. 9A is a dose-response curve of G6P enzyme- α expression induced upon infection with rAAV from the DTC161 vector. FIG. 9B is a dose-response curve of G6P enzyme- α expression induced upon infection with a rAAV from the DTC177 vector (represented by SEQ ID NO: 1). The X-axis represents rAAV dose used to infect HuH7 hepatocytes. The Y-axis represents induced G6PC mRNA expression in the cells. rAAV derived from the development of mass-produced DTC161 vector was used as a reference standard. Abbreviations used: REF-reference standard; UNK-test sample.

Detailed Description

The present invention provides a series of novel agents and compositions for therapeutic applications. The molecules and compositions of the invention are useful for ameliorating, preventing or treating a disease associated with glycogen storage disease type Ia (GSD-Ia) or increasing the presence or function of glucose-6-phosphatase-alpha (G6P enzyme-alpha) in a subject.

Unless otherwise indicated, technical terms are conventionally used. Definitions of terms commonly used in molecular biology can be found in: benjamin Lewis, Genes V [ Gene V ], Oxford university Press, 1994(ISBN 0-19-854287-9); kendrew et al (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd, 1994(ISBN 0-632-02182-9); and Robert a. meyers (ed.), Molecular Biology and Biotechnology a Comprehensive Desk Reference [ Molecular Biology and Biotechnology: integrated desk reference, published by VCH publishing company (VCH Publishers, Inc.), 1995(ISBN 1-56081-.

For ease of reviewing the various embodiments of the present disclosure, the following explanation of specific terms is provided:

adeno-associated virus (AAV): a small, replication-defective, non-enveloped virus infects humans and some other primates. AAV is known not to cause disease and to elicit a very mild immune response. Gene therapy vectors using AAV can infect dividing and quiescent cells and persist extrachromosomally without integrating into the genome of the host cell. These characteristics make AAV an attractive viral vector for gene therapy. There are currently 12 recognized AAV serotypes (AAV 1-12).

Application (Administration/administerer): an agent, such as a therapeutic agent (e.g., a recombinant AAV), is provided or administered to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, cholangioluminal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

Codon-optimized: a "codon-optimized" nucleic acid refers to a nucleic acid sequence that has been altered such that codons are optimal for expression in a particular system (e.g., a particular species or group of species). For example, the nucleic acid sequence may be optimized for expression in mammalian cells or in a particular mammalian species (e.g., human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.

Enhancer: a nucleic acid sequence which increases the rate of transcription by increasing the activity of a promoter.

G6 PC: the gene located on human chromosome 17q21, encoding glucose-6-phosphatase- α (G6P enzyme- α). G6P enzyme-. alpha.is a 357 amino acid hydrophobin with 9 helices anchoring it in the endoplasmic reticulum (Chou et al, Nat Rev Endocrinol [ Nature review Endocrinol ]6:676 688, 2010). The G6P enzyme-alpha protein catalyzes the hydrolysis of glucose 6-phosphate to glucose and phosphate at the final stages of gluconeogenesis and glycogenolysis and is a key enzyme in glucose homeostasis. Mutation of the G6PC gene results in glycogen storage disease type Ia (GSD-Ia), a metabolic disorder characterized by severe fasting hypoglycemia associated with the accumulation of glycogen and fat in the liver and kidney.

Glycogen Storage Disease (GSD): a group of diseases caused by defects in the synthesis or breakdown of glycogen in muscle, liver and other tissues. GSD may be either genetic or acquired. Genetic GSD is caused by any congenital metabolic error involved in these processes. There are currently 11 recognized glycogen storage diseases (GSD I, II, III, IV, V, VI, VII, IX, XI, XII and XIII). GSD-I consists of two autosomal recessive genetic disorders GSD-Ia and GSD-Ib (Chou et al, Nat Rev Endocrinol [ Nature review Endocrinol ]6:676-688, 2010). GSD-Ia is caused by a deficiency in glucose-6-phosphatase-alpha. Defects in the glucose-6-phosphate transporter (G6PT) are responsible for GSD-Ib.

Glycogen storage disease type Ia (GSD-Ia): GSD-Ia, also known as hepatic glycogen accumulation, is the most common glycogen storage disease with an incidence of about 1 in 100,000 live births. GSD-Ia is a genetic disease caused by a deficiency in glucose-6-phosphatase-alpha (G6P enzyme-alpha). A deficiency in the G6P enzyme- α impairs the liver's ability to produce free glucose from glycogen and gluconeogenesis. Patients affected by GSD-Ia failed to maintain glucose homeostasis and developed fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia (Chou et al, Nat Rev Endocrinol [ Nature review Endocrinol ]6:676-688, 2010). GSD-Ia cannot be cured at present.

An intron: a piece of DNA in which the gene does not contain information encoding a protein. Introns are removed prior to translation of messenger RNA.

Inverted Terminal Repeat (ITR): a symmetric nucleic acid sequence in the genome of the adeno-associated virus required for efficient replication. The ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as origins of replication for viral DNA synthesis and are required for vector encapsidation.

Separating: an "isolated" biological component (e.g., a nucleic acid molecule, protein, virus, or cell) has been substantially separated or purified away from other biological components in a cell or tissue of an organism or the organism itself (where the components naturally occur, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, and cells). Nucleic acid molecules and proteins that have been "isolated" include those purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.

Operatively connected to: a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

A pharmaceutically acceptable carrier: pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by e.w. martin, Mack Publishing co., Easton, Pa.,15th Edition (1975) describe compositions and formulations suitable for drug delivery of one or more therapeutic compounds, molecules or agents.

In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral formulations typically comprise injectable fluids, which include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like as vehicles. For solid compositions, such as in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

Prevention, treatment or amelioration of diseases: "preventing" a disease (e.g., GSD-Ia) refers to inhibiting the overall progression of the disease. "treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it begins to progress. "improvement" refers to a reduction in the number or severity of signs or symptoms of disease.

A promoter: a region of DNA that directs/initiates transcription of a nucleic acid (e.g., a gene). Promoters include the necessary nucleic acid sequences adjacent to the transcription start site.

Purification of: the term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is a peptide, protein, virus, or other active compound that is completely or partially separated from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to peptides, proteins, viruses, or other active compounds that have been separated from cells, cell culture media, or other crude preparations and fractionated to remove various components (e.g., proteins, cell debris, and other components) of the initial preparation.

And (3) recombination: a recombinant nucleic acid molecule is a nucleic acid molecule having a sequence that does not occur naturally or has a sequence that is made by artificially combining two otherwise separate segments of sequence. Such artificial combination may be achieved by chemical synthesis or by artificial manipulation of isolated fragments of the nucleic acid molecule, for example by genetic engineering techniques.

Similarly, a recombinant virus is a virus that comprises sequences (e.g., genomic sequences) that are not naturally occurring or that have been made by artificial combination of sequences from at least two different sources. The term "recombinant" also includes nucleic acids, proteins and viruses that have been altered by the addition, substitution or deletion of only a portion of a native nucleic acid molecule, protein or virus. As used herein, "recombinant AAV" refers to an AAV particle in which a recombinant nucleic acid molecule, e.g., a recombinant nucleic acid molecule encoding G6P enzyme- α, is packaged.

Sequence identity: identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of identity or similarity between the sequences. Sequence identity can be measured in percent identity; the higher the percentage, the more identical the sequence. Sequence similarity can be measured in terms of percent similarity (taking into account conservative amino acid substitutions); the higher the percentage, the more similar the sequence. Homologues or orthologues of nucleic acid or amino acid sequences have a relatively high degree of sequence identity/similarity when aligned using standard methods. When orthologous proteins or cdnas are derived from more closely related species (e.g., human and mouse sequences), this homology is more pronounced compared to more closely related species (e.g., human and nematode sequences).

Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in: smith & Waterman, adv.appl.math. [ applied math progression ]2:482,1981; needleman & Wunsch, J.mol.biol. [ journal of molecular biology ]48:443,1970: Pearson & Lipman, Proc.Natl.Acad.Sci.USA [ Proc.Natl.Acad.Sci ]85:2444,1988; higgins & Sharp, Gene [ Gene ],73:237-44, 1988; higgins & Sharp, CABIOS5:151-3, 1989; corpet et al, Nuc. acids Res. [ nucleic acid research ]16:10881-90, 1988; details of the sequence alignment method and homology calculation are given in Huang et al Computer applications in the Biosciences 8, 155-.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J.mol.biol. [ journal of molecular biology ]215: 403-. More information can be found on the NCBI website.

Serotype: a group of closely related microorganisms (e.g., viruses) is distinguished by a characteristic antigen group.

Filling sequence: refers to a nucleotide sequence contained within a larger nucleic acid molecule (e.g., a vector) that is typically used to create a desired spacing between two nucleic acid features (e.g., between a promoter and a coding sequence) or to extend a nucleic acid molecule to a desired length. The stuffer sequence contains no protein coding information and may be of unknown/synthetic origin and/or unrelated to other nucleic acid sequences within the larger nucleic acid molecule.

Subject: living multicellular vertebrate organisms, this category including human and non-human mammals.

The synthesis comprises the following steps: generated in the laboratory by artificial means, for example, synthetic nucleic acids can be chemically synthesized in the laboratory.

A therapeutically effective amount of: an amount of a particular drug or therapeutic agent (e.g., recombinant AAV) is sufficient to achieve a desired effect in a subject or cell treated with the agent. The effective amount of the agent will depend on several factors, including but not limited to the subject or cell being treated, and the mode of administration of the therapeutic composition.

Carrier: a vector is a nucleic acid molecule that allows for the insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector may include a nucleic acid sequence, such as an origin of replication, that allows it to replicate in a host cell. The vector may also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of the inserted gene or genes. In some embodiments herein, the vector is an AAV vector.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an", "the" include plural referents unless the context clearly dictates otherwise. "comprising A or B" means including A, or B, or A and B. It is further understood that all base sizes or amino acid sizes and all molecular weights or molecular weight values given for a nucleic acid or polypeptide are approximate and provided for illustration. Although methods and materials similar or equivalent to those disclosed herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

I. Recombinant nucleic acid

One aspect of the invention provides a recombinant nucleic acid sequence comprising a modified G6PC promoter/enhancer (GPE) lacking one or more Alu elements as compared to a wild-type GPE, wherein the modified GPE is capable of directing expression of an encoding gene encoding G6P enzyme- α (SEQ ID NO: 5). In some embodiments, the modified GPE is obtained by removing one or more Alu elements from the endogenous promoter of the human G6PC gene, for example, one or more of the contiguous nucleotides 1079-. In some other embodiments, the modified GPE is obtained by removing one or more Alu elements from the endogenous promoter of the G6PC gene of other mammals, e.g., non-human primates, sheep, rodents, etc. In some embodiments, the modified GPE does not affect G6PC gene expression compared to wild-type GPE. In some embodiments, the modified GPE is capable of enhancing expression of the G6PC gene compared to a wild-type GPE. In some embodiments, the modified GPE has comparable activity to wild-type GPE driving expression of the G6PC gene in the liver, and very low activity driving expression of the G6P enzyme-a in other tissues.

In some embodiments, a modified GPE comprises a nucleic acid sequence that lacks a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to contiguous nucleotide 1079-1272(Alu-1) from SEQ ID NO: 6. In some embodiments, a modified GPE comprises a nucleic acid sequence that lacks a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to contiguous nucleotides 1633-1968(Alu-2) from SEQ ID NO: 6. In some embodiments, a modified GPE comprises a nucleic acid sequence that lacks a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to contiguous nucleotides 2140-2495(Alu-3) from SEQ ID NO: 6.

In some embodiments, a modified GPE comprises a nucleic acid sequence that lacks a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to Alu-1 and a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to Alu-2 from SEQ ID No. 6. In some embodiments, a modified GPE comprises a nucleic acid sequence that lacks a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to Alu-1 and a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to Alu-3 from SEQ ID No. 6. In some embodiments, a modified GPE comprises a nucleic acid sequence that lacks a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to Alu-2 and a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to Alu-3 from SEQ ID No. 6. In some embodiments, a modified GPE comprises a nucleic acid sequence that lacks a sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to an Alu-1 sequence, a sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to Alu-2, and a sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, (wt.)% identical to Alu-3 from SEQ ID No. 6, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the same sequence.

In some embodiments of the invention, the recombinant nucleic acid sequence comprises a GPE having a nucleic acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to contiguous nucleotide 146-2123 of SEQ ID NO:1, wherein the GPE is capable of directing expression of a coding sequence encoding the G6P enzyme- α. In some embodiments, the recombinant nucleic acid sequence comprises a GPE having a nucleic acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID No. 7, wherein the GPE is capable of directing expression of a coding sequence encoding G6P enzyme-a. In some embodiments, the recombinant nucleic acid sequence comprises a GPE having a nucleic acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID No. 8, wherein the GPE is capable of directing expression of a coding sequence encoding G6P enzyme- α. In some embodiments, the recombinant nucleic acid sequence comprises a GPE having a nucleic acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID No. 9, wherein the GPE is capable of directing expression of a coding sequence encoding G6P enzyme-a. In some embodiments, the recombinant nucleic acid sequence comprises a GPE having a nucleic acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO 10, wherein the GPE is capable of directing expression of a coding sequence encoding G6P enzyme-a. In some embodiments, the recombinant nucleic acid sequence comprises a GPE having a nucleic acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID No. 11, wherein the GPE is capable of directing expression of a coding sequence encoding G6P enzyme- α. In some embodiments, the recombinant nucleic acid sequence comprises a GPE having a nucleic acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO 12, wherein the GPE is capable of directing expression of a coding sequence encoding G6P enzyme-a.

Another aspect of the invention provides a recombinant nucleic acid sequence comprising a modified GPE disclosed herein and a coding sequence encoding G6P enzyme- α. In some embodiments, G6P enzyme-a includes an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID No. 5. In some embodiments, the G6P enzyme- α comprises SEQ ID No. 5 or an active fragment or variant thereof. In an exemplary embodiment, the G6P enzyme- α comprises or consists of SEQ ID No. 5.

In some embodiments, the coding sequence encoding G6P enzyme- α incorporates a nucleic acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID No. 4.

In some embodiments, the coding sequence encoding human G6P enzyme- α is a codon optimized for expression in human cells. The human G6PC cDNA can be codon optimized using OptimumGene codon optimization technology (Kinsry, GenScript), Piscataway (Piscataway, N.J.). The optimized G6PC cDNA sequence can be examined and further modified to eliminate potential Alternative Reading Frames (ARFs) from the internal non-frame ATG sequence which theoretically can encode peptides of 9 or more amino acids in length. For example, the codon-optimized G6PC cDNA sequence may be further modified to avoid potentially cytotoxic T lymphocyte responses to the transgenic products produced by ARF (Li et al, 2009, PNAS [ Proc. Natl. Acad. Sci. USA ]106: 10770-4). In some embodiments, the codon-optimized coding sequence for human G6P enzyme- α incorporates a nucleic acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID No. 3.

In some embodiments, a recombinant nucleic acid sequence comprising a modified GPE as described herein and a coding sequence encoding G6P enzyme- α further comprises an intron and/or a polyadenylation signal. In some embodiments, the intron is located between the GPE and G6P enzyme- α coding sequences. In some embodiments, the intron is a chimeric intron that increases G6P enzyme- α transgene expression. The intron may be composed of a5 '-donor site from the first intron of the human β -globin gene and a branch and 3' -acceptor site from the intron of the heavy chain variable region of the immunoglobulin gene, where the sequences of the donor and acceptor sites and the branch point site have been altered to match the consensus sequence for splicing. In some embodiments, an intron comprises a nucleic acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO 13.

A polyadenylation signal may be placed downstream of the coding sequence encoding the G6P enzyme- α to effect polyadenylation of the G6PC mRNA. A variety of polyadenylation signals may be used, for example the simian virus 40(SV40) late polyadenylation signal, the hGH polyadenylation signal, the BGH polyadenylation signal or the rbGlob polyadenylation signal. In some embodiments, the polyadenylation signal is the SV40 late polyadenylation signal. In some embodiments, the polyadenylation signal comprises a nucleic acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID No. 14.

Another aspect of the invention provides a recombinant vector comprising a modified GPE and a coding sequence encoding the G6P enzyme- α disclosed herein. In some embodiments, the recombinant vector further comprises an intron and/or a polyadenylation signal as described herein. The vector may be a mammalian expression vector, a bacterial expression vector, a yeast expression vector, a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, an RNAi vector, a Cre-Lox expression vector, a CRISPR expression vector, a TALEN expression vector, or the like. The vector may further comprise an intron and/or a polyadenylation signal as described herein. In some embodiments, the recombinant vector further comprises a stuffer nucleic acid sequence located between the GPE and the intron and/or between the intron and the G6P enzyme- α coding sequence.

In some embodiments, the recombinant vector is an AAV vector. The AAV vector can be any of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12) as well as AAV vectors isolated from human and non-human primate tissue in any of more than 100 variants (see, e.g., Choi et al, Curr Gene Ther. [ current Gene therapy ],5: 299-. Any serotype of AAV vector can be used in the present invention, and the selection of AAV serotype will depend in part on the cell type or cell types targeted by the gene therapy. For treatment of GSD-Ia, the liver is one of the relevant target organs.

In some embodiments, the recombinant AAV vector comprises AAV ITR sequences that function as both an origin of replication for vector DNA and as packaging signals for the vector genome when AAV and adenoviral helper functions are provided in trans. In addition, ITRs are targets for single strand endonucleases of large Rep proteins, splitting a single genome from replicative intermediates.

In some exemplary embodiments, the AAV vector is an AAV serotype 8(AAV8) vector, and the vector includes a modified GPE, an intron, a coding sequence encoding G6P enzyme-a, and an SV40 late polyadenylation signal described herein. In some embodiments, the vector further comprises two AAV2 Inverted Terminal Repeat (ITR) sequences (SEQ ID NO: 15): the 5 'one of GPE and the 3' one of polyadenylation signal. In some specific non-limiting embodiments, the recombinant vector comprises a nucleic acid sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID No. 1 or SEQ ID No. 2.

Host cells comprising recombinant nucleic acids

Further provided are isolated host cells comprising the recombinant nucleic acid molecules or vectors disclosed herein. A variety of host cells may be used, such as bacterial, yeast, insect, mammalian cells, and the like. In some embodiments, the host cell may be a cell (or cell line) suitable for production of recombinant AAV (rAAV), such as HeLa, Cos-7, HEK293, A549, BHK, Vero, RD, HT-1080, ARPE-19, or MRC-5 cells.

The recombinant nucleic acid molecule or vector can be delivered to the host cell culture using any suitable method known in the art. In some embodiments, a stable host cell line is produced having a recombinant nucleic acid molecule or vector inserted into its genome. In some embodiments, a stable host cell line is produced comprising an AAV vector described herein. Following transfection of AAV vectors into host cultures, integration of rAAV into the host genome can be determined by a variety of methods, such as antibiotic selection, fluorescence-activated cell sorting, Western blotting, PCR-based detection, fluorescence in situ hybridization, e.g., Nakai et al, Nature Genetics [ Nature Genetics ] (2003)34, 297-302; philpott et al, Journal of Virology (2002)76(11):5411-5421 and Howden et al, J Gene Med [ Gen. Pharma ] 2008; 10: 42-50. In addition, stable cell lines can be established according to protocols well known in the art, for example, in Clark, Kidney International [ International journal of the Kidney ] Vol.61 (2002): S9-S15, and Yuan et al, Human Gene Therapy [ Human Gene Therapy ] 5 months 2011; 22(5) 613-24.

Recombinant AAV

The invention also provides rAAV comprising an AAV capsid and an AAV vector genome as described herein. The AAV capsid may be serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or rh 10). In some embodiments, the capsid is an AAV8 capsid.

rAAV can be produced by a host cell that has the AAV vector and AAV Rep and Cap gene functions disclosed herein, as well as additional helper functions. Rep and Cap gene functions can be provided to the host cell in a variety of ways, for example, by plasmids or any type of vector comprising wild-type AAV Rep and Cap genes, and electroporation of Rep and Cap mRNA. Additional helper functions may be provided, for example, by Adenovirus (AV) infection, a plasmid carrying all of the required AV helper function genes, or other viruses such as Herpes Simplex Virus (HSV) or baculovirus. Any gene, gene function, or other genetic material necessary for production of rAAV by a host cell may be transiently present within the host cell, or stably inserted into the host cell genome. rAAV production methods suitable for use in the methods of the invention include those disclosed in: clark et al, Human Gene Therapy [ Human Gene Therapy ]6: 1329-.

In an exemplary embodiment, HEK293 cells were transfected with: an AAV vector comprising the nucleic acid sequence of SEQ ID NO 1 or SEQ ID NO 2; plasmids encoding the four wild-type AAV2 viral replication (Rep) proteins and the three wild-type AAV viral capsid (cap) proteins from serotype 8; and plasmids containing regions of the adenoviral genome important for AAV replication (i.e., E2A, E4, and VA RNA). rAAV containing the AAV8 capsid can then be produced and isolated from the host cell. In some embodiments, the modified GPE lacking one or more Alu elements in the AAV vector enhances packaging of the rAAV produced from the host cell. In some embodiments, a modified GPE lacking one or more Alu elements in an AAV vector affects self-complementary structure formation and thus increases production of rAAV produced from the host cell.

Lysis of AAV-infected cells can be accomplished by methods that chemically or enzymatically treat the cells to release infectious viral particles. These methods include the use of nucleases (e.g., benzonase or dnase), proteases (e.g., trypsin), or detergents or surfactants. Physical disruption, such as homogenization or grinding, or application of pressure by a microfluidizer pressure unit, or freeze-thaw cycling may also be used. Alternatively, supernatants can be collected from AAV-infected cells without cell lysis.

It may be desirable to purify the sample containing the rAAV and helper viral particles to remove, for example, cell lysis productsRaw cell debris. Minimal purification methods for helper virus and AAV particles are known in the art, and any suitable method can be used to prepare a sample comprising AAV and helper virus particles for use in the methods of the invention. Two exemplary purification methods are cesium chloride (CsCl) based and iodixanol based density gradient purification. Strobel et al, Human Gene Therapy Methods]26(4) 147-. Affinity chromatography using, for example, AVB Sepharose affinity resin (GE Healthcare Bio-Sciences AB), Uppsala (Uppsala), Sweden) or POROS may also be used^TM CaptureSelect^TMAAV8, AAV9, or AAVX affinity resins (Thermo Fisher Scientific, miller burg, pa) to accomplish minimal purification. AAV purification Methods using AVB Sepharose affinity resin are described, for example, in Wang et al, Mol Ther Methods Clin Dev [ molecular therapy-Methods and clinical development ]],2:15040(2015)。

Inactivation of the helper virus by heat may be required. The heat inactivation technique is based on the different thermal stability of AAV and helper virus particles. For example, AAV particles can be heated to temperatures up to 56 ℃ and remain intact, while AV particles become inactivated. Conway et al, Gene Therapy]6,986-993,1999 describes the differential heat inactivation of HSV in samples containing AAV. Heat inactivation may be accomplished by any known method. In the examples described below, thermal inactivation is achieved using a thermal cycler to rapidly heat and cool a sample volume of 300 μ L or less. This system was chosen because it relies on primarily conductive heat transfer, making it a viable model for both continuous flow systems and larger volume systems employing active mixing. Examples of continuous flow systems include passing the sample through a continuous flow heat exchanger, such as DHX for biotherapeutic manufacturing^TMDisposable heat exchangers (seimer feishell technologies, miller sburg, pa). Such a system allows an operator to control the heat inactivation process by controlling the flow rate of the sample through the heat exchanger, thereby controlling the duration of the heating process and the temperature of the heat exchanger, therebyThe temperature of the heat inactivation is controlled.

Alternatively, thermal inactivation may be achieved using batch systems of various sizes. For example, heat inactivation can be accomplished on a 1L scale by placing the sample containing AAV into a 1L PETG vial and placing the vial in a water bath set to the desired inactivation temperature for the desired time, and mixing; for example, the sample may be heated to 47 ℃ for 20 minutes. On a larger scale, heat inactivation may be achieved by placing the rAAV-containing sample into a 5L bioprocessing bag on a temperature controlled rocking platform set at the desired inactivation temperature for a desired time. For example, the rocking platform may be set at 49 ℃, a rocking speed of 30RPM, and a mixing angle of 12 ° for 40 minutes.

Heat inactivation can occur at any temperature, where there is sufficient difference in stability between the rAAV particle and the helper viral particle that the helper viral particle is substantially inactivated while the active rAAV particle remains. Those skilled in the art will appreciate that higher temperatures may be required to achieve greater levels of AV reduction. In some embodiments, the heat-inactivation step comprises using a buffer containing a kosmotropic salt and/or a divalent or trivalent cation. WO/2017/172772 describes a method for heat inactivation in the presence of a buffer containing a kosmotropic salt and/or a divalent or trivalent cation.

Once heat inactivation is complete, it may be necessary or desirable to determine the inactivation efficiency. The efficacy of the inactivation protocol is determined by an assay that detects the presence of replication competent helper virus, such as a plaque assay. Plaque assays for helper viruses are well known to those skilled in the art and include plaque assays for AV, HSV, baculovirus, and the like. Plaque assays for adenoviruses can be performed using any suitable cell type, such as HeLa or HEK293 cells. Standard plaque assay Protocols are described, for example, in Current Protocols in Human Genetics],2003. Alternative assays for measuring adenovirus titers include methods that allow for the identification of infected cells in culture by detecting viral proteins (such as hexon protein) using immunocytochemical staining. Such assays include QuickTiter^TMAdenovirus titer immunoassay kit (Cell Biolabs, san Diego)Ca). Inactivation efficiency is usually reported as Log Reduction of Virus (LRV).

Quantification of rAAV particles is complicated by the fact that AAV infection does not lead to cytopathic effects in vitro, and therefore plaque assays cannot be used to determine infectious titers. However, quantification of AAV particles can be performed using a variety of methods, including quantitative polymerase chain reaction (qPCR) (Clark et al, hum. Gene Ther. [ human Gene therapy ]10, 1031-. DNase Resistant Particles (DRP) can be quantified by real-time quantitative polymerase chain reaction (qPCR) (DRP-qPCR) in a thermal cycler (e.g., iCycler iQ 96-well modular thermal cycler (Bio-Rad, Hercules, Calif.) in the presence of DNase I (100U/ml; Promega, Madison, Wis.) samples containing AAV particles are incubated at 37 ℃ for 60 minutes, followed by proteinase K (Invitrogen, Calsbad), Calif.) digestion (10U/ml) for 60 minutes at 50 ℃ and then denaturation at 95 ℃ for 30 minutes using primer-probe sets that are specific for non-native portions of the AAV vector genome, e.g., poly (A) sequences of the protein of interest, The length and composition of the probes and amplification sequences, and any suitable set of cycling parameters can be used to amplify the PCR products. Alternatives are disclosed in, for example, Lock et al, Human Gene Therapy Methods [ Human Gene Therapy Methods ]25(2):115-125 (2014).

TCID may be used₅₀(50% tissue culture infectious dose) assay to determine infectivity of rAAV particles, e.g., in Zhen et al, Human Gene Therapy]15: 709-. In this assay, AAV vector particles are serially diluted and used to co-infect Rep/Cap expressing cell lines in 96-well plates with AV particles. At 48 hours post-infection, total cellular DNA was extracted from the infected and control wells. Replication of AAV vectors was then measured using qPCR with transgene-specific probes and primers. TCID₅₀Infectivity per ml (TCID)₅₀Per ml) useThe equation calculates the ratio of AAV positive wells using 10-fold serial dilutions.

Recombinant AAV for gene therapy

AAV belongs to the Parvoviridae (Parvoviridae) and the Dependovirus (Dependovirus). AAV is a small, non-enveloped virus that packages a linear, single-stranded DNA genome. Both the sense and antisense strands of AAV DNA are packaged into the AAV capsid at the same frequency.

The AAV genome is characterized by two Inverted Terminal Repeats (ITRs) that flank two open reading frames (ORBs). For example, in the AAV2 genome, the first 125 nucleotides of an ITR are palindromes, which fold upon themselves to maximize base pairing and form a T-hairpin structure. The other 20 bases of the ITR, called the D sequence, remain unpaired. ITRs are cis-acting sequences important for AAV DNA replication; the ITR is the origin of replication and serves as a primer for the synthesis of the second strand by the DNA polymerase. The double stranded DNA formed during this synthesis, called replicative monomers, is used for the second round of self-initiated replication and forms replicative dimers. These double-stranded intermediates are processed by a strand displacement mechanism to produce single-stranded DNA for packaging and double-stranded DNA for transcription. Located within the ITRs are Rep binding elements and terminal dissociation sites (TRSs). These features are used by the viral regulatory protein Rep to process double-stranded intermediates during AAV replication. In addition to their role in AAV replication, ITRs are essential for AAV genomic packaging, transcription, negative regulation under non-permissive conditions, and site-specific integration (Days and Berns, Clin Microbiol Rev [ review in clinical microbiology ]21(4): 583-.

The left ORF of AAV comprises a Rep gene, which encodes four proteins-Rep 78, Rep68, Rep52, and Rep 40. The right ORF contains the Cap gene, which produces three viral capsid proteins (VP1, VP2, and VP 3). The AAV capsid comprises 60 viral capsid proteins arranged in icosahedral symmetry. VP1, VP2 and VP3 were present in a molar ratio of 1:1:10 (Daya and Berns, Clin Microbiol Rev [ review of clinical microbiology ]21(4): 583-.

AAV is one of the most commonly used viruses in gene therapy today. While AAV infects humans and some other primates, it is known not to cause disease and elicits a very mild immune response. Gene therapy vectors using AAV can infect dividing and quiescent cells and persist extrachromosomally without integrating into the genome of the host cell. Due to the advantageous characteristics of AAV, the present disclosure contemplates the use of AAV in the recombinant nucleic acid molecules and methods disclosed herein.

AAV has several desirable characteristics of gene therapy vectors, including the ability to bind to and enter target cells, enter the nucleus, be expressed in the nucleus for long periods of time, and low toxicity. However, the small size of the AAV genome limits the size of heterologous DNA that can be incorporated. To minimize this problem, AAV vectors have been constructed that do not encode Rep and Integration Efficiency Elements (IEEs). ITRs are retained because they are cis signals required for packaging (Daya and Berns, Clin Microbiol Rev [ clinical microbiology review ],21(4): 583-.

Methods for producing rAAV suitable for Gene therapy are well known in the art (see, e.g., U.S. patent application Nos. 2012/0100606; 2012/0135515; 2011/0229971; and 2013/0072548; and Ghosh et al, Gene Ther [ Gene therapy ]13(4):321-329,2006), and may be used with the recombinant nucleic acid molecules and methods disclosed herein.

The present disclosure provides compositions comprising the rAAV disclosed herein and a pharmaceutically acceptable carrier. Suitable pharmaceutical formulations for administering rAAV can be found, for example, in U.S. patent application publication No. 2012/0219528. Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by e.w. martin, Mack Publishing co., Easton, Pa.,15th Edition (1975) describe compositions and formulations suitable for drug delivery of one or more therapeutic compounds, molecules or agents.

In some embodiments, the rAAV is formulated in a buffer/carrier suitable for infusion into a human subject. The buffer/carrier should include components that prevent the rAAV from adhering to the infusion tube, but do not interfere with the binding activity of the rAAV in vivo. Various suitable solutions may include one or more of the following: buffered saline, surfactant and a physiologically compatible salt or mixture of salts whose ionic strength is adjusted to be equal to about 100mM sodium chloride (NaCl) to about 250mM sodium chloride, or a physiologically compatible salt adjusted to an equal ionic concentration. The pH may be in the range of 6.5 to 8.5, or 7 to 8.5, or 7.5 to 8. Suitable surfactants or combinations of surfactants may be selected from poloxamers, i.e. non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene 10 (poly (propylene oxide)) and two pendant hydrophilic chains of polyoxyethylene (poly (ethylene oxide)), SOLUTOL HS 15(Macrogol-15 hydroxystearate), LABRASOL (glyceryl polyoxyoctanoate), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol and polyethylene glycol.

The invention also provides methods of treating a subject diagnosed with glycogen storage disease type 1a (GSD-Ia) and administering to the subject a therapeutically effective amount of a rAAV disclosed herein (or a composition comprising a rAAV).

The rAAV or rAAV-containing compositions described herein can be administered using any suitable method or route. Routes of administration include, for example, systemic, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. In some embodiments, the rAAV or composition comprising the rAAV is administered intravenously.

The particular dose administered may be a uniform dose for each patient, e.g., 1.0x10¹³-1.0x10¹⁵Individual viral Genome Copies (GC)/patient. Alternatively, the dosage of the patient may be adjusted according to the approximate weight or surface area of the patient. Other factors in determining an appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient. Further refinement of the calculations required to determine an appropriate therapeutic dose is routinely made by those skilled in the art, particularly in light of the dosage information and determinations disclosed herein. Dosage may also be determined by using known assays for determining dosage in combination with appropriate dose-response numbersIs determined accordingly. For example, the glucose may be assessed by assessing the first occurrence of a hypoglycemic event (defined as controlling the fasting period (which will end when hypoglycemia occurs or up to 15 hours)<60mg/dL(<3.33mmol/L)) to determine the optimal biological dose of rAAV administered in minutes. When monitoring the progression of the disease, the dosage of the individual patient may also be adjusted.

In some embodiments, the rAAV is administered, e.g., at about 1.0x10¹¹One genome copy per kilogram patient body weight (GC/kg) to about 1x10¹⁴GC/kg, about 5X10¹¹One genome copy per kilogram patient body weight (GC/kg) to about 5x10¹³GC/kg, or about 1X10¹²To about 1x10¹³Dosage of Gc/kg, as measured by qPCR or digital droplet PCR (ddPCR). In some embodiments, the rAAV is administered at about 2 × 10¹²GC/kg dose was administered. In some embodiments, the rAAV is administered at about 6 x10¹²GC/kg dose was administered. In some embodiments, the rAAV is administered at about 1 × 10¹³GC/kg dose was administered. rAAV may be administered in a single dose or in multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses) as desired for a desired therapeutic outcome.

The dose may be administered one or more times per week, month or year, even once every 2 to 20 years. For example, each dose may be administered at least 1 week apart, 2 weeks apart, 3 weeks apart, one month apart, 3 months apart, 6 months apart, or 1 year apart. One of ordinary skill in the art can readily estimate the repetition rate of administration based on the measured residence time and the concentration of the targetable construct or complex in the body fluid or tissue.

Methods for increasing recombinant virus yield and gene therapy efficacy

The invention also provides a method of increasing recombinant viral yield of a host cell, wherein the method comprises removing one or more Alu elements or Alu element-associated sequences from a recombinant viral vector. In some embodiments, the Alu element-related sequence is at least 50% identical to an Alu element (e.g., contiguous nucleotides 1079-. The recombinant viral vector may be, for example, a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector. In some embodiments, one or more Alu elements or Alu element-associated sequences are removed from the promoter/enhancer region within the viral vector. In some embodiments, one or more Alu elements or Alu element-related sequences are removed from an intron region within the viral vector. In some embodiments, removal of one or more Alu elements or Alu element-associated sequences reduces self-complementing structures formed during packaging of the recombinant viral particles, thereby increasing viral yield from the host cell.

Throughout the specification, where a composition is described as having, including, or comprising a particular compound, or where processes and methods are described as having, including, or comprising a particular step, it is contemplated that additionally there is a composition of the invention consisting essentially of, or consisting of, the recited compound, and a process and method according to the invention consisting essentially of, or consisting of, the recited processing step.

In the present application, when an element or component is referred to as being included in and/or selected from a list of recited elements or components, it is understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from the group consisting of two or more of the recited elements or components.

In addition, it should be understood that elements and/or features of the compositions or methods described herein may be combined in various ways, whether explicit or implicit herein, without departing from the spirit and scope of the invention. For example, when a particular compound is referred to, unless otherwise understood from the context, the compound may be used in various embodiments of the compositions of the invention and/or in the methods of the invention. In other words, in this application, embodiments have been described and depicted in a manner that enables writing and drawing of clear and concise applications, but it is intended and will be understood that embodiments may be combined or separated in various ways without departing from the teachings of this application and one or more inventions. For example, it should be understood that all of the features described and depicted herein may be applicable to all aspects of one or more of the inventions described and depicted herein.

It should be understood that unless otherwise understood from context and usage, the expression "at least one" includes each of the listed objects individually as well as various combinations of two or more of the listed objects after the expression. The expression "and/or" in relation to three or more of the listed objects shall be understood to have the same meaning unless otherwise understood from the context.

It will be understood that the use of the terms "include, include", "have, have", "contain, contain" and "containing", including grammatical equivalents thereof, are intended to be open ended and non-limiting, e.g., does not exclude additional unrecited elements or steps unless expressly stated otherwise or understood from the context.

If the term "about" is used before a numerical value, the invention also includes the particular numerical value itself unless specifically stated otherwise. As used herein, unless otherwise specified or inferred, the term "about" refers to a ± 10% variation of the nominal value.

It should be understood that the order of steps or order of performing certain actions is immaterial so long as the invention remains operable. Further, two or more steps or actions may be performed simultaneously.

The use of any and all examples, or exemplary language, such as "for example" or "including" herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Examples

The invention being generally described will now be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the invention, and are not intended to limit the invention.

Example 1 AAV vectors and rAAV produced from the vectors

AAV vectors

An AAV vector comprising the G6PC expression cassette surrounded by two AAV2 inverted terminal repeats (ITR, SEQ ID NO:15) was constructed. The G6PC expression cassette is bounded at its 5 'end by Yiu et al, 2010, Molecular Therapy [ Molecular Therapy ]18(6):1076-84 primer sequence "1S" and its associated KpnI restriction endonuclease site, at the 5' end of the G6PC promoter/enhancer (GPE). By alignment with the SV40 genome and the associated SalI restriction endonuclease site, the G6PC expression cassette was bounded at the 3' end by its SV40 late polyadenylation signal. All G6PC expression cassettes contained GPE, introns, codon optimized human G6PC gene and SV40 late poly a tail, as shown in fig. 1A. Different versions of the G6PC expression cassette were generated, each version containing either wild-type GPE or modified GPE, as shown in figure 1B. The elements of the G6PC expression cassette are described below.

The wild-type G6PC promoter/enhancer (GPE, SEQ ID NO:6) is from homo sapiens and is defined by RefSeq NG _ 011808. The sequence is an endogenous promoter of a human G6PC gene, and has almost unique activity in the liver and extremely low activity in the kidney. The wild-type GPE comprises 3 Alu elements located at the consecutive nucleotides 1079-1272(Alu-1), 1633-1968(Alu-2) and 2140-2495(Alu-3) of SEQ ID NO: 6. AAV vector DTC161 comprises a G6PC expression cassette, which comprises wild-type GPE. The AAV vector DTC175 comprises a G6PC expression cassette, said G6PC expression cassette comprising a GPE in which Alu-1 and Alu-2 sequences are deleted. AAV vector DTC176 comprises a G6PC expression cassette comprising a GPE in which the orientation of Alu-1 and Alu-2 is reversed, the G6PC expression cassette. AAV vector DTC177 (represented by SEQ ID NO: 1) comprises the G6PC expression cassette, said G6PC expression cassette comprising GPE in which Alu-1, Alu-2 and Alu-3 sequences are deleted. AAV vector DTC178 contains the G6PC expression cassette, which G6PC expression cassette comprises GPE in which Alu-3 sequences are deleted. AAV vector DTC179 comprises a G6PC expression cassette comprising a GPE in which the orientation of the Alu-3 sequence is reversed, the G6PC expression cassette.

The chimeric intron (SEQ ID NO:13) is composed of a5 '-donor site derived from the first intron of the human β -globin gene and a branch and 3' -acceptor site derived from the intron of the heavy chain variable region of the immunoglobulin gene. The sequences of the donor and acceptor sites as well as the branch point site have been altered to match the consensus sequence for splicing (CI-neo Mammarian Expression Vector Technical Bulletin [ CI-neo Mammalian Expression Vector Technical Bulletin ] TB215, Promega Life Sciences Corporation [ Promega Life Sciences Corporation ]). The purpose of the chimeric intron is to increase gene expression.

The G6PC cDNA (SEQ ID NO:4) was from homo sapiens and was codon optimized for expression in human cells. Proprietary OptimumGene can be used^TMCodon optimization techniques (Kinsry (GenScript), Piscataway (Piscataway, N.J.) codon-optimized the human G6PC cDNA. The optimized cDNA sequence can be examined and further modified to eliminate potential Alternative Reading Frames (ARFs) from the internal non-frame ATG sequence which theoretically can encode peptides of 9 or more amino acids in length. For example, the codon optimized G6PC cDNA is represented by SEQ ID NO 3.

The simian virus 40(SV40) late polyadenylation signal (Genbank # J02400, SEQ ID NO:14) provides a cis sequence for efficient polyadenylation of the G6PC mRNA. This element serves as a signal for a specific cleavage event at the 3' end of the nascent transcript and the addition of a long poly A tail.

Each G6PC expression cassette was cloned into an AAV vector. All AAV vectors have a backbone encoding a kanamycin resistance gene. AAV vector DTC161(pdtx. hg6pcco.401) is shown as an example in fig. 2.

rAAV virions

The AAV vector genome is a single-stranded DNA genome. Only sequences between and including ITR sequences are packaged into AAV virions. Viral particles were produced by transfection of three plasmids into human embryonic kidney 293(HEK293) cells, which provided the E1a and E1b gene products. The first plasmid is an AAV vector as described herein. The second plasmid is pAAV2-8.KanR (p2123-FH), a packaging plasmid containing the wild-type AAV2 rep and AAV8 cap genes. The third plasmid is pAdDeltaF6(Kan), which is a helper adenovirus plasmid.

The gland-associated Rep/Cap plasmid pAAV2/8, KanR (p2123-FH) (8354bp) encodes four wild-type AAV2 viral replication (Rep) proteins and three wild-type AAV VP capsid (Cap) proteins from serotype 8. A schematic of the pAAV2/8.KanR (p2123-FH) plasmid is shown in FIG. 3. In the plasmid, the AAV p5 promoter that normally drives expression of the Rep gene has moved from the 5 'end of the Rep region to the 3' end of the AAV8 cap region. This arrangement introduces a spacer (i.e., a plasmid backbone) between the promoter and the Rep gene, resulting in down-regulation of Rep expression and an increase in the ability to support high titer rAAV production. Both the kanamycin resistance gene and the MB1 origin were included for plasmid production in E.coli.

Plasmid pAdDeltaF6(Kan) contains regions of the adenoviral genome important for AAV replication, namely E2A, E4 and VARNA (fig. 4). Adenovirus E1 function is also essential, but is provided by HEK293 host cells. The plasmid does not contain other adenoviral replication, structural genes, or cis elements essential for adenoviral replication, such as the adenoviral ITRs, and therefore, it is not expected that infectious adenovirus will be produced. Both the kanamycin resistance gene and the MB1 origin were included for plasmid production in E.coli.

Example 2 Alu element deletion increases rAAV yields

AAV vectors containing wild-type GPE or modified GPE were used to transfect HEK293 cells with the Rep/Cap and helper plasmids described above.

The Alu element was either deleted in the modified GPE (in the DTC175, DTC177 and DTC178 vectors) or the Alu element was reversed in orientation (in the DTC176 and DTC179 vectors). The DTC175 viral vector comprises a G6PC expression cassette, said G6PC expression cassette comprising a GPE in which the Alu-1 and Alu-2 sequences are deleted, the DTC176 viral vector comprises a G6PC expression cassette, said G6PC expression cassette comprising a GPE in which all three Alu elements are present but the orientation of the Alu-1 and Alu-2 sequences is reversed, the DTC177 viral vector comprises a G6PC expression cassette, said G6PC expression cassette comprising a GPE in which the Alu-1, Alu-2 and Alu-3 sequences are deleted, the DTC178 viral vector comprises a G6PC expression cassette, said G6PC expression cassette comprising a GPE in which the Alu-3 sequences are deleted, and the DTC179 viral vector comprises a G6PC expression cassette, said G6PC expression cassette comprising a GPE in which all three Alu elements are present but the orientation of the Alu-3 sequences is reversed.

On day 5 after co-transfection, infected cells were incubated in the presence of sodium deoxycholate andin lysis buffer (2) at 37 ℃ for 2 hours. The sample supernatant is then digested sequentially with dnase I and proteinase K to release rAAV genomic DNA. The BGH-polya coding region of AAV vectors was then amplified using TaqMan qPCR to determine rAAV genome copy number (GC) from rAAV plasmid standard curves. Fig. 5 is a bar graph showing rAAV titers produced from host cells following transfection with various AAV vectors. As such, fig. 5 shows rAAV titers measured by qPCR and indicates that deletion of one or more Alu elements from GPE (in DTC175, DTC177, and DTC178 vectors) significantly increased rAAV yield compared to DTC161, which comprises wild-type GPE. However, reversing one or more Alu elements (in the DTC176 and DTC179 vectors) did not improve rAAV production compared to DTC 161. Quantification of virus yield is summarized in table 1. As shown in table 1, deletion of one or more Alu elements from GPE can significantly improve rAAV production. A second analysis showing rAAV titers produced plotted as a function of vector genome size is presented as the graph in figure 6. As shown in FIG. 6, DTC177 (represented by SEQ ID NO: 1) which is the vector genome with the smallest size produced the highest titer.

Table 1: quantitative summary of viral yield produced by HEK293 cells following transfection with AAV vectors (DTC161, DTC175, DTC176, DTC177, DTC178 or DTC 179).

Example 3-Alu element deletion improves rAAV packaging

To assess whether deletion of one or more Alu elements affects packaging of raavs, raavs produced as described in example 2 were harvested and total DNA was isolated from each rAAV (produced from control viral vectors DTC161, DTC175, DTC176, DTC177, DTC178, or DTC 179). About 7.12x10¹⁰The total amount of each rAAV GC was subjected to agarose gel electrophoresis, and then stained with SYBR Gold. The control viral vector used in this experiment was AAV8-LSP-hFIXco3-WPRE-pA (produced in Willok, Virovek, custom purified, catalog/lot No. 061015, 150282),it provides for known DNA size migration on gels and confirms that experimental methods can disrupt AAV capsid integrity and release packaged DNA.

As shown in FIG. 7, the total viral DNA was between 3.8kb and 5 kb. "" indicates the complete genome of full length DNA isolated from control viral vectors after capsid degradation and treatment with Sodium Dodecyl Sulfate (SDS).

Higher staining intensity of full-length DNA isolated from rAAV lacking one or more Alu elements DTC177, DTC175 and DTC178 in GPE was observed (see figure 7). This result indicates that deletion of at least one Alu element can increase rAAV packaging of the full-length viral genome. In addition, ultracentrifuge traces for DTC161 and DTC177 particles analyzed for empty (occurring at about 60S) and particles containing full vector DNA (occurring at about 100S), indicating that the construct lacking the Alu element produced a higher percentage of intact particles, respectively (see fig. 8A-8B). Fig. 8A is a graph showing analytical ultracentrifugation traces of particle density from DTC161 vector preparations produced in HEK293 cells. FIG. 8B is a graph showing analytical ultracentrifugation traces from particle density of DTC177 vector (represented by SEQ ID NO: 1) preparations produced in HEK293 cells.

These data indicate that improved packaging occurs in vectors lacking Alu sequences.

Example 4 deletion of Alu element in GPE does not affect promoter efficacy

To assess whether deletion of one or more Alu elements affected the efficacy of GPE to direct G6PC gene expression in vivo, HuH7 liver cells were infected with rAAV derived from different vectors (as shown in table 2). 48 hours after infection, cells were lysed and total mRNA was harvested. The G6PC mRNA expression was analyzed by quantitative RT-PCR. FIGS. 9A-9B show exemplary dose response curves between rAAV dose and G6PC mRNA expression levels in HuH7 hepatocytes for rAAV derived from DTC161 and DTC177 (represented by SEQ ID NO: 1). Fig. 9A is a dose-response curve of G6P enzyme- α expression induced upon infection with rAAV containing DTC 161. FIG. 9B is a dose-response curve of G6P enzyme- α expression induced upon infection with a rAAV containing DTC177 (represented by SEQ ID NO: 1). To calculate the relative potency of the test samples, the RNA values (number of genomic copies/. mu.g total RNA) at each multiplicity of infection (MOI) for the reference standard (rAAV derived from DTC161 vector produced in development lots) and the test samples were added to the GraphPad Prism file template. The template uses a log-log transformation of the data and a linear fit that is constrained so that the curves have a shared slope. The Y-axis corresponds to the RNA value and the X-axis corresponds to the MOI of the sample. The Y-intercept and slope data generated by the GraphPad Prism template was used to determine the X-intercept of the reference standard and test sample by implementing the following formula: x Int ═ (0- [ Y intercept ])/slope. The final relative efficacy value is determined by comparing the X-intercepts using the following formula: 10 { [ X intercept reference ] - [ X intercept sample ].

Table 2 summary of the relative potency of GPE in each rAAV sample.

Sample (I)	Relative potency
		DTC-161	80％
DTC-175	83％
		DTC-176	79％
DTC-177	89％
		DTC-178	75％
DTC-179	85％

This result indicates that the deletion of one or more Alu elements in GPE (in the DTC175, DTC177 and DTC178 vectors) does not compromise the efficacy of GPE in driving G6PC gene expression in vivo.

All publications, patents, and documents mentioned specifically herein are incorporated by reference for all purposes.

It is to be understood that this invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be covered only by the appended claims.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Likewise, the terms "a" (or "an"), "one or more (one or more)" and "at least one (atleastone)" may be used interchangeably herein. It should also be noted that the terms "comprising", "containing", "including" and "having" may be used interchangeably.

Without further elaboration, it is believed that one skilled in the art can, based on the description above, utilize the present invention to its fullest extent. The specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose.